I worked in the laboratory of the late professor Alan C. Sartorelli, where we had a long standing history of designing agents to selectively target tumor cells in hypoxic see this article. Article Physicochemical Considerations of Tumor Selective Drug Deliv...

Our laboratory around the time of its shutdown (due to our PI's death) operated on a shoe string budget, thus we had to come up with our own cheap methodologies to produce the conditions that mimicked the hypoxic oxygen concentrations found in solid tumors. Since, these conditions were necessary for the activation our prodrugs. Thus we came up with a cheap method to achieve these goals.

Article Generation of Oxygen Deficiency in Cell Culture Using a Two-...

Article A Simple and Inexpensive Method to Control Oxygen Concentrat...

It would be possible to use variations of such methods to produce steady-state oxygen concentrations to mimic other physiological, and disease state oxygen concentrations.

Good luck with your research endeavors.

p.s. I've been retired from active laboratory research since early 2017, but have continued to publish the odd paper.

Hello,

Thanks for the great discussion.

We are using a perfusion bioreactor system with O2 sensors upstream and downstream from the media flow. I have also seen O2 sensors placed in the middle of scaffolds in 3D cell cultures for perfusion systems.

Thank you Philip G Penketh for the interesting references about creating hypoxia for tumor cell culturing. The method sounds really useful. If I read it correctly, 2D cell cultures were used. Had you also tried the method with tumor spheroids?

Hello Genesis Marrero

thanks for the insight into your work. Was the upstream/downstream measurement of oxygen for your questions already sufficient or were there also some considerations to measure directly at/inside the scaffold? Or is that not possible due to the experimental setup?

Hi Christoph, we didn't use 3d spheroids, only 2d cultures or cell suspensions, using 3d spheroids would likely add another oxygen concentration gradient. Therefore the oxygen concentration set by the enzyme system would represent the highest oxygen concentration for the peripheral cells, and the inner cells would experience greater hypoxia.

It is obvious when using bath cultures in suspension that the cell density is tightly dependent upon oxygen supply that means of the coefficient tranfer between oxygen bubbling and its solubility in the medium. The rotation speed of the helicoidal device maintaining the cells in suspension is a crucial point : increasing the rpm enhances oxygen suppliy but also creates shear stress to the cells, it is thus important to obtain the better compromise

We believe thebupstream/downstream O2 concentrations were sufficient, since cells grew as expected. We have not tried to put an O2 sensor in the central channel. Our experimental setup is not designed for that.

I agree with Phillip's comment on the hypoxia at the core of 3D cultures. This will be diffusion dependent, determined largely by the size of the organoids/spheroids and will be very difficult to measure in vitro.

I've not come across genuinely perfused 3D cultures yet, which would be another way to control oxygen concentration.

Perhaps an empirical approach could be taken by IF staining for oxidative stress markers for example.

Oxygen measurement in 3D cell cultures is a critical aspect that directly impacts cellular behavior and experimental outcomes. The choice between hypoxia, hyperoxia, and physioxia conditions depends on the research objectives and the physiological relevance you aim to mimic.

Hypoxia, characterized by lower oxygen levels, is commonly used to simulate conditions in various disease states such as solid tumors. This oxygen deprivation can help study cellular responses to limited oxygen availability, which is often linked to altered gene expression and cellular metabolism.

Hyperoxia, on the other hand, involves exposing cells to higher-than-normal oxygen concentrations. This approach is valuable for investigating oxidative stress, wound healing, and the effects of oxygen toxicity. However, it's important to note that hyperoxia might not always reflect physiological conditions accurately.

Physioxia, also known as normoxia, aims to replicate the oxygen levels in healthy tissues within the body. This approach better mimics cells' natural environment and allows for a more accurate representation of their behavior under standard conditions.

The choice between these conditions should be based on the specific research questions you're addressing. While hypoxia and hyperoxia offer insights into extreme scenarios, physioxia conditions are often preferred when aiming to understand cellular behavior under normal physiological conditions.

Ultimately, the decision should be guided by the goals of your study and the relevance of the chosen oxygen environment to the biological context you are investigating.

Here is a figure, which illustrates the range of oxygen concentrations that can be found in various tissues, you can find more details in the below linked publication.

Article Physicochemical Considerations of Tumor Selective Drug Deliv...

Hi Christoph Grün, these are all very good points and note my comment comes from a place where I'm chatting with researchers often who are navigating 3D cell models (especially organoids) where everything is often quite new.

I do think with 3D models there is still a good amount of research that demonstrates/suggests that providing the 3D models an "in vivo like" microenvironment provides better translatability in general but often does not often account for O2 differences thoughout the model which tend to be higher on the surface and a lower value on the inside which makes sense physiologically.

https://www.oxford-optronix.com/resources/5-reasons-to-control-oxygen-in-organoid-models

Now good news for 3D model researchers is there do seem to be technologies that are being utilized to garner some of this important information. We have seen a few teams utilize our OxyLite sensor placed into their 3D models or constructs to provide some real-time oxygen data but as the sensor has a 250um diameter these do need to be rather large 3D models. Outside of that I have come across some techniques that seem to provide this type of data data but cannot speak to how well those work or their general feasibility to the average research group.

Article Direct Measurements of Oxygen Gradients in Spheroid Culture ...

Article Oxygen diffusion in ellipsoidal tumour spheroids

Article Dispersible oxygen microsensors map oxygen gradients in thre...

I think the important takeaway is moving forward, year over year, there should be more data on the differences of the external and internal pO2 of these cellular constructs (based on several parameter's) which should help guide future researcher understanding without the full need to take the measurements.